Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Details

Serval ID
serval:BIB_1C5AE8384F97
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.
Journal
European Journal of Biochemistry
Author(s)
Nguyen V.T., Baker D.P., Tricot C., Baur H., Villeret V., Dideberg O., Gigot D., Stalon V., Haas D.
ISSN
0014-2956 (Print)
ISSN-L
0014-2956
Publication state
Published
Issued date
1996
Volume
236
Number
1
Pages
283-293
Language
english
Abstract
Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.
Keywords
Allosteric Regulation, Amino Acid Sequence, Bacterial Proteins/genetics, Bacterial Proteins/metabolism, Base Sequence, Enzyme Stability, Escherichia coli Proteins, Membrane Proteins/genetics, Membrane Proteins/metabolism, Molecular Sequence Data, Mutagenesis, Ornithine Carbamoyltransferase/genetics, Ornithine Carbamoyltransferase/metabolism, Protein Conformation, Protein Kinases, Pseudomonas aeruginosa/enzymology, Recombinant Fusion Proteins/metabolism, Structure-Activity Relationship
Pubmed
Web of science
Create date
25/01/2008 17:01
Last modification date
20/08/2019 12:52
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